The family of Hox genes encodes a set of homeodomain containing transcriptional regulators that are necessary for the development of all metazoans. Expression of Hox genes along the antero-posterior axis is necessary to set the genetic program that defines cell- and tissue-identity during development; however misexpression and misregulation of Hox genes has been strongly linked to oncogenesis. The absence of a complete understanding of the molecular mechanisms of Hox-dependent transcriptional regulation, though, hampers our ability to design cancer therapeutics targeted against these factors. The long term objective of this proposal is to better understand these mechanisms by specifically addressing the hypothesis that Hox transcriptional regulators define cell-fate through a specification of genome architecture, resulting in wide-spread gene expression changes. As alterations in genome architecture have also been correlated with oncogenesis, this proposal has the potential to provide insight into the multi-level process that, when altered, has the capacity to initiate and maintain the abnormal gene expression programs that promote cancer-cell formation. To test this hypothesis, the studies proposed here will focus on the development of the wing and haltere in Drosophila melanogaster, which are derived from serially homologous larval imaginal discs that differ only in the expression of a single Hox gene, Ultrabithorax (Ubx). Ubx expression in the haltere is necessary and sufficient to alter the genetic program that would otherwise result in the formation of the wing, as mutations in Ubx result in four-winged flies. Expanding upon a novel technology developed in the sponsor's laboratory, termed Cell- and Gene-specific Chromatin Immunoprecipitation (cgChIP), I will assay the 3D genomic architecture of regulatory regions associated with genes at many levels of the genetic hierarchy that promote haltere formation in response to Ubx. Specifically, I will ask whether any differences in genomic architecture exist in the wing versus the haltere at regions in the Ubx locus that function to maintain Ubx expression in the ON/OFF state and at Ubx-target genes. Having established that differences exist at these loci, I will determine if Ubx expression and activity is necessary to establish the genome architecture found in the haltere, thus providing mechanistic insight into how Ubx, and other Hox genes, define cell-fate. The evolutionary conservation of Hox genes throughout metazoans allows for the fundamental insights gained from this work in Drosophila to be translated into an understanding of Hox-dependent specification of genome architecture in higher organisms, and ultimately into the discovery of novel targets for cancer therapeutic intervention.